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Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex


We constructed error-correcting DNA barcodes that allow one run of a massively parallel pyrosequencer to process up to 1,544 samples simultaneously. Using these barcodes we processed bacterial 16S rRNA gene sequences representing microbial communities in 286 environmental samples, corrected 92% of sample assignment errors, and thus characterized nearly as many 16S rRNA genes as have been sequenced to date by Sanger sequencing.

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Figure 1: Operation of Hamming error-correcting codes.
Figure 2: UniFrac clustering by community was essentially perfect with sequences from pyrosequencing.


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We thank N. Pace, L. Gold and F. Accurso for support and encouragement, J.I. Gordon and R. Bushman for helpful discussions, and R. Ley, C. Lozupone and D. McDonald for feedback on the manuscript. This work was supported in part by the US National Institutes of Health–University of Colorado at Boulder Molecular Biophysics Training Program (T32GM065103), and grants from the Cystic Fibrosis Foundation and National Institutes of Health (U01 HL081335-01, P01DK078669).

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Authors and Affiliations



M.H. and R.K. designed and implemented the analyses, and wrote the manuscript. J.J.W., J.K.H. and N.J.G. generated the 454 dataset.

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Correspondence to Rob Knight.

Supplementary information

Supplementary Text and Figures

Supplementary Figure 1, Supplementary Data 1, Supplementary Methods (PDF 422 kb)

Supplementary Data 2

Decoding software example, Readme file and demo output (PDF 67 kb)

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Hamady, M., Walker, J., Harris, J. et al. Error-correcting barcoded primers for pyrosequencing hundreds of samples in multiplex. Nat Methods 5, 235–237 (2008).

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